There are many instances where the efficacy of a therapeutic protein is limited by an unwanted immune reaction to the therapeutic protein. Several mouse monoclonal antibodies have shown promise as therapies in a number of human disease settings but in certain cases have failed due to the induction of significant degrees of a human anti-murine antibody (HAMA) response (Schroff, R. W. et al. (1985) Cancer Res. 45: 879-885; Shawler, D. L. et al. (1985) J. Immunol. 135: 1530-1535). For monoclonal antibodies, a number of techniques have been developed in attempt to reduce the HAMA response (WOA8909622; EPA0239400; EPA0438310; WOA9106667; EPA0699755). These recombinant DNA approaches have generally reduced the mouse genetic information in the final antibody construct whilst increasing the human genetic information in the final construct to result in antibody molecules which are generally termed “humanised” antibodies.
Humanised antibodies, for the most part, are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or primate, and this process is sometimes termed “CDR grafting”. Generally additional Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues and in some instances other substitutions are made to further restore the antibody function. Typically humanised antibodies are reconstituted into whole antibody molecules comprising two variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanised antibody will also generally comprise at least a portion of a human derived immunoglobulin constant region (Fc) (Jones et al. (1986), Nature 321: 522-525; Reichmann et al. (1988), Nature 332: 323-329). Notwithstanding, humanised antibodies have, in several cases, still elicited an immune response in patients (Issacs J. D. (1990) Sem. Immunol. 2: 449, 456; Rebello, P. R. et al. (1999) Transplantation 68: 1417-1420).
Key to the induction of an immune response is the presence within the protein of peptides that can stimulate the activity of T-cells via presentation on MHC Class II molecules, so-called “T-cell epitopes”. Such T-cell epitopes are commonly defined as any amino acid residue sequence with the ability to bind to MHC Class II molecules. Implicitly, a “T-cell epitope” means an epitope which when bound to MHC molecules can be recognized by a T-cell receptor (TCR), and which can, at least in principle, cause the activation of these T-cells by engaging a TCR to promote a T-cell response.
MHC Class II molecules are a group of highly polymorphic proteins which play a central role in helper T-cell selection and activation. The human leukocyte antigen group DR (HLA-DR) are the predominant isotype of this group of proteins and isotypes HLA-DQ and HLA-DP perform similar functions. In the human population, individuals bear two to four DR alleles, two DQ and two DP alleles. The structure of a number of DR molecules has been solved and these appear as an open-ended peptide binding groove with a number of hydrophobic pockets which engage hydrophobic residues (pocket residues) of the peptide (Brown et al. Nature (1993) 364: 33; Stern et al. (1994) Nature 368: 215). Polymorphism identifying the different allotypes of Class II molecule contributes to a wide diversity of different binding surfaces for peptides within the peptide binding grove and at the population level ensures maximal flexibility with regard to the ability to recognize foreign proteins and mount an immune response to pathogenic organisms.
An immune response to a therapeutic protein proceeds via the MHC Class II peptide presentation pathway. Here exogenous proteins are engulfed and processed for presentation in association with MHC Class II molecules of the DR, DQ or DP type. MHC Class II molecules are expressed by professional antigen presenting cells (APCs), such as macrophages and dendritic cells amongst others. Engagement of a MHC Class II peptide complex by a cognate T-cell receptor on the surface of the T-cell, together with the cross-binding of certain other co-receptors such as the CD4 molecule, can induce an activated state within the T-cell. Activation leads to the release of cytokines further activating other lymphocytes such as B cells to produce antibodies or activating T killer cells as a full cellular immune response.
T-cell epitope identification is the first step to epitope elimination. Identified epitopes may be eliminated by judicious amino acid substitution or other modification strategies. Such an approach is recognized in WO98/52976 and WO00/34317 where in the latter case computational threading techniques are described as a means to identify polypeptide sequences with the potential to bind a sub-set of human MHC Class II DR allotypes and the predicted T-cell epitopes are removed by amino acid substitution within the protein of interest.
It would be desirable to identify and to remove, or at least to reduce, T-cell epitopes from a given, in principle therapeutically valuable, but originally immunogenic peptide, polypeptide or protein. One of these therapeutically valuable molecules is a monoclonal antibody with binding specificity for the human B-cell antigen CD20. The preferred monoclonal antibodies of the present invention are modified forms of the antibody 2B8 and Leu16. Antibody 2B8 is described in U.S. Pat. No. 5,736,137, the disclosure of which is incorporated herein by reference. Antibody Leu16 is described in Wu et al. Protein Engineering (2001) 14:1025-1033, the disclosure of which is incorporated herein by reference.
CD20 is a non-glycosylated phosphoprotein of 35,000 Daltons, typically designated as the human B lymphocyte restricted differentiation antigen Bp35B. The protein is a highly cell specific surface molecule expressed on pre-B and mature B-cells including greater than 90% of B-cell non-Hodgkin's lymphomas (NHL). Monoclonal antibodies and radioimmunoconjugates targeting CD20 have emerged as new treatments for NHL. The most significant example includes the parental antibody of the present invention, namely monoclonal antibody 2B8 (Reff, M. E. et al. (1994) Blood 83: 435-445). The variable region domains of 2B8 have been cloned and combined with human constant region domains to produce a chimeric antibody designated C2B8 which is marketed as RITUXAN™ in the U.S.A. or MABTHERA® (rituximab) in Europe. C2B8 is recognized as a valuable therapeutic agent for the treatment of NHL and other B-cell diseases (Maloney, D. G. et al. (1997) J. Clin. Oncol. 15: 3266-3274; Maloney, D. G. et al. (1997) Blood 90: 2188-2195).
An additional example of an anti-CD20 therapeutic is provided by the antibody B1, described in U.S. Pat. No. 6,090,365, the disclosure of which is incorporated herein by reference. This antibody has similarly achieved registration for use as a NHL therapeutic although in this case the molecule (BEXXAR™) is a 131I radioimmunoconjugate. The native B1 (non-conjugated) antibody has utility in ex vivo purging regimens for autologous bone marrow transplantation therapies for lymphoma and refractory leukaemia (Freedman, A. S. et al. (1990), J. Clin. Oncol. 8: 784).
Despite the success of antibodies such as C2B8 (rituximab) and BEXXAR™ there is a continued need for anti-CD20 analogues with enhanced properties. There is a particular need for enhancement of the in vivo characteristics when administered to the human subject. In this regard, it is highly desired to provide anti-CD20 antibodies with reduced or absent potential to induce an immune response in the human subject. Such proteins would display an increased circulation time within the human subject and would be of particular benefit in chronic use settings such as is the case for the therapeutic use of anti-CD20 molecules. The present invention provides modified anti-CD20 antibodies that display a relatively low level of immunogenicity in vivo.